Assay for identifying antibody producing cells

ABSTRACT

The present invention provides a homogeneous assay for identifying an antibody producing cell producing an antibody which binds to a selected antigen comprising: a) providing a population of antibody producing cells; b) incubating said population of antibody producing cells with a selected antigen and a labeled anti-antibody antibody, wherein said anti-antibody antibody is capable of distinguishing cells producing an antibody which binds to the selected antigen from those cells which do not; and c) identifying an antibody producing cell capable of producing an antibody which binds to the selected antigen.

The present invention relates generally to improved methods forproducing antibodies and more specifically provides a homogeneous assayfor obtaining antibodies.

The selected lymphocyte antibody method (SLAM) for generating monoclonalantibodies overcomes the limitations of both hybridoma technology andbacterially expressed antibody libraries by enabling high affinityantibodies generated during in vivo immune responses to be isolated fromany species (Babcook et al., 1996, Proc. Natl. Acad. Sci, 93,7843-7848). SLAM enables a single lymphocyte that is producing anantibody with a desired specificity or function to be identified withina large population of lymphoid cells and the genetic information thatencodes the specificity of the antibody to be rescued from thatlymphocyte. Antibody producing cells which produce antibodies which bindto selected antigens are detected using an adapted hemolytic plaqueassay method (Jerne and Nordin, 1963, Science, 140, 405). In this assayerythrocytes are coated with the selected antigen and incubated with thepopulation of antibody producing cells and a source of complement.Single antibody producing cells are identified by the formation ofhemolytic plaques. Plaques of lysed erythrocytes are identified using aninverted microscope and the single antibody producing cell of interestat the centre of the plaque is removed using micromanipulationtechniques and the antibody genes from the cell are cloned by reversetranscription PCR. Other methods for detecting single antibody-producingcells of a desired function have already been described in InternationalPatent Specification, WO 92/02551.

In the hemolytic plaque assay described above the red blood cells aretypically coated with antigen via a biotin/streptavidin coupling systemthat requires the antigen to be biotinylated. This method is thereforerestricted to antigens that are available in a pure form and to thosethat can be biotinylated without affecting epitope presentation. Thismethod clearly precludes the isolation of antibodies against a widerange of antigens. For example, many proteins are difficult to purify,particularly cell surface expressed proteins, such as type III proteins.Many proteins alter their conformation and presentation of desirableeptiopes upon biotinylation, for example proteins that contain lysinegroups in their active site.

It may also be desirable to produce antibodies against unknown antigens,such as proteins expressed on the surface of cells, such as tumor cells.The direct use of tumor cells in the plaque assay instead of antigencoated erythrocytes is difficult to achieve given the requirement forcell lysis to occur in order for plaques contaning antibody producingcells to be identified. Cell lysis is dependent on cell type, antigenand antibody concentration. Red blood cells coated with the desiredantigen will bind large amounts of available antibody and will lysereadily in the presence of complement. Other cell types such as tumorcells will not lyse so readily, especially when the availability ofantigen on the surface may be very low and hence antibody binding willbe low.

The current invention addresses these difficulties by providing improvedmethods for producing antibodies and more specifically by providing ahomogeneous assay for obtaining antibodies. This improved assay has manyadvantages over the methods described above, allowing the identificationof antibodies that bind to any antigen, including unknown antigens, cellsurface antigens and antigens which cannot be biotinylated withoutaltering the presentation of desirable epitopes. As a result, antibodieswith binding specificities that were previously unidentifiable byconventional plaque assays can now be produced. In addition the assay ismore facile than the hemolytic plaque assay and antibody producing cellscan be identified more quickly.

We have demonstrated that it is possible to obtain antibody producingcells producing antibodies which bind to an antigen by incubating apopulation of antibody producing cells with an antigen source in thepresence of labeled antibodies which bind to the antibodies produced bythe antibody producing cells. Suprisingly, it is possible to distinguishthose cells producing antibodies which bind to an antigen over thosewhich do not without the need for wash steps to remove unbound label.Thus, according to the present invention, there is provided ahomogeneous assay for identifying an antibody producing cell producingan antibody which binds to a selected antigen comprising:

-   a) providing a population of antibody producing cells;-   b) incubating said population of antibody producing cells with a    selected antigen and a labeled anti-antibody antibody, wherein said    anti-antibody antibody is capable of distinguishing cells producing    an antibody which binds to the selected antigen from those cells    which do not; and-   c) identifying an antibody producing cell producing an antibody    which binds to the selected antigen.    The term ‘antibody’ as used herein includes any recombinant or    naturally occurring immunoglobulin molecule such as a member of the    IgG class e.g. IgG1 and also any antigen binding immunoglobulin    fragment, such as Fv, Fab′ and F(ab′)₂ fragments, and any    derivatives thereof, such as single chain Fv fragments.

The term ‘antibody producing cell’ as used herein means any cell capableof secreting an antibody, such as a B-lymphocyte, a plasma cell, aplasmablast, an activated B cell or a memory B cell. Antibody-producingcells for use in the invention may be obtained from an animal which haseither been immunized with an antigen, or which has developed an immuneresponse to an antigen as a result of disease. Other antibody producingcells for use in the present invention may include any transformed cellin particular, any mammalian cells which express immunoglobulin genes orparts thereof. In one example the populations of antibody producingcells for use in the present invention produce a range of antibodieswith different binding specificities.

The assay of the present invention may also be used to identify highyielding antibody producing cells from a population of antibodyproducing cells which all produce the same antibody. The term ‘highyielding’ as used herein refers to antibody producing cells that produceantibodies of a known specificity but for which it would be desirable toidentify those cells producing the antibody most efficiently.Identification of the high yielding cell will allow the cell to beisolated and clonally reproduced. In one example the high yieldingantibody producing cell is a hybridoma cell. In another example the highyielding antibody producing cell is a transformed cell in particular, amammalian cell which expresses immunoglobulin genes or parts thereof.Examples of such mammalian cells include but are not limited to NS0,CHO, COS and 293 cells.

The term ‘antigen’ as used herein refers to any known or unknownsubstance that can be recognised by an antibody, including proteins,glycoproteins and carbohydrates. Preferably these antigens includebiologically active proteins, such as hormones, cytolines, and theircell surface receptors, bacterial or parasitic cell membranes orpurified components thereof, and viral antigens. In one example theantigen is available in a pure form obtained either by directpurification from the native source or by recombinant expression andpurification of said antigen. Preferably the purified antigen is coupledto erythrocytes or any other particle such as a bead for incorporationinto the assay. In another example the antigen is one which is difficultto purify, such antigens include but are not limited to cell surfaceexpressed proteins such as receptors, particularly type III proteins. Inanother example the presentation of desirable epitopes on the antigen isaltered upon biotinylation, this includes but is not limited to proteinswhich contain lysines in their active site regions. In another examplethe antigen may be expressed on the surface of a cell, either naturallyor recombinantly. Such cells may include but are not limited tomammalian cells, immunomodulatory cells, lymphocytes, monocytes,polymorphs, T cells, tumor cells, yeast cells, bacterial cell,infectious agents, parasites, plant cells, transfected cells such asNS0, CHO, COS, 293 cells. In one example the antigens expressed on thesurface of said cells are antigens which are difficult to purify orantigens which lose desired epitopes upon biotinylation such as thoseantigens described above.

In another example the antigen is a cell or a population of cells forwhich it would be desirable to isolate antibodies to, such as mammaliancells, immunomodulatory cells, lymphocytes, monocytes, polymorphs, Tcells, tumor cells, yeast cells, bacterial cell, infectious agents,parasites, and plant cells. In one embodiment the cell is a tumor cell.

The term ‘homogeneous assay’ as used herein refers to an assay wherebyall components of the assay are combined together to identify antibodyproducing cells without the need to remove unbound labeled anti-antibodyantibodies. The term ‘labeled anti-antibody antibody’ refers to labeledantibodies which bind to any region of the antibodies produced by theantibody producing cells, regardless of the binding specificity of thoseantibodies. Preferably said anti-antibody antibodies are from onespecies while the antibody producing cells are from another. Preferablythese antibodies bind to the Fc portion of the antibody produced by theantibody producing cell.

The labeled anti-antibody antibodies are capable of distinguishing cellsproducing antibodies that bind to the selected antigen from those cellsthat do not. Appropriate labels are well known in the art and caninclude but are not limited to chemiluminescence, enzyme and fluorescentlabels. Preferably the label is a fluorescent label. The fluorescentlabel conjugated to the anti-antibody antibodies can be any fluorescentlabel including but not limited to Aqua, Texas-Red, FITC, rhodamine,rhodamine derivative, fluorescein, fluorescein derivative, cascade blue,Cy5 and phycoerythrin. Preferably the fluorescent conjugate is FITC.Thus in one particular example of an assay according to the presentinvention, the antibody producing cells are from rabbits and the labeledanti-antibody antibodies are fluorescent labeled goat anti-rabbitanti-Fc antibodies.

In the assay of the present invention the antibody producing cellsproducing antibodies which bind to the selected antigen aredistinguished from those that do not by detecting the increasedconcentration of labeled anti-antibody antibodies surrounding saidcells. Preferably this is achieved by visualising the labeledanti-antibody antibodies and hence the antibody producing cellsurrounded by said antibodies. In one example this is achieved using amicroscope. Preferably the label is detected using an invertedmicroscope with a mercury vapour UV lamp and a filter set appropriatefor the conjugate used. Preferably the filter set is a fluoresceinfilter set. Thus in one example, where the label is a fluorescent labelthe antibody producing cells producing antibodies which bind to theselected antigen are identified by a localised increase in fluorescencesurrounding said cells. The present invention also provides a method ofproducing an antibody which binds to a selected antigen comprising:

-   a) providing a population of antibody producing cells;-   b) incubating said population of antibody producing cells with a    selected antigen and a labeled anti-antibody antibody, wherein said    anti-antibody antibody is capable of distinguishing cells producing    an antibody which binds to the selected antigen from those cells    which do not;-   c) identifying an antibody producing cell producing an antibody    which binds to the selected antigen;-   d) isolating the identified antibody producing cell; and optionally-   e) synthesizing an antibody therefrom.    Where desired, steps (d) and (e) can be repeated more than once to    isolate more than one antibody producing cell and to synthesize more    than one antibody. The present invention therefore extends to at    least one antibody producing cell identified by the above method and    at least one antibody synthesized from said cell(s).

Antibody producing cells identified using the homogeneous assaydescribed herein are isolated directly from the assay usingmicromanipulation techniques well known in the art.

Antibodies can be synthesized from the isolated antibody producing celleither directly or indirectly. Direct synthesis can be achieved byculturing the isolated antibody producing cell in an appropriate medium.Indirect synthesis can be achieved by isolating the genes encoding theantibodies or parts thereof and expressing them in a host cell usingmethods well known in the art. A vector containing the antibody gene(s)is transfected into a host cell and the host cell cultured in anappropriate medium such that the antibody or antibody fragment with thedesired specificity is produced in the host cell.

DETAILED DESCRIPTION OF THE ASSAY

Antibody-producing cells for use in the present invention may beobtained from any appropriate source, including an animal which haseither been immunized with a selected antigen, or which has developed animmune response to an antigen as a result of disease.

Animals may be immunized with a selected antigen using any of thetechniques well known in the art suitable for generating an immuneresponse (see Handbook of Experimental Immunology, D. M. Weir (ed.), Vol4, Blackwell Scientific Publishers, Oxford, England, 1986). Manywarm-blooded animals, such as humans, rabbits, mice, rats, sheep, cowsor pigs may be immunized in order to obtain antibody-producing cells.However, mice, rabbits and rats are generally preferred.

High numbers of antibody producing cells can be found in the spleen andlymph node of the immunised animal and once an immune response has beengenerated and the animal has been sacrificed, the spleen and lymph nodesare removed. A single cell suspension of antibody producing cells isprepared using techniques well known in the art. Antibody producingcells can also be obtained from an animal that has generated the cellsduring the course of a disease. For instance, antibody producing cellsfrom a human with a disease of unknown cause, such as cancer, may beobtained and used to assist in the identification of antibodies whichhave an effect on the disease process or which may lead toidentification of an agent or body component that is involved in thecause of the disease. Similarly, antibody-producing cells may beobtained from subjects with disease of known cause such as malaria orAIDS. These antibody producing cells may be derived from the blood orlymph nodes, as well as from other diseased or normal tissues.

Antibody producing cells may also be obtained by culture techniques suchas in vitro immunization. Examples of such methods are described by C.R. Reading in Methods in Enzymology 121:18-33 (J. J. Langone, H. H. vanVunakis (eds,), Academic Press Inc., N. Y.). Antibody producing cellsmay also be obtained from very early monoclonal or oligoclonal fusioncultures produced by conventional hybridoma technology. The populationof antibody producing cells may be enriched for use in the assay bymethods based upon the size or density of the antibody producing cellsrelative to other cells. An example of the use of Percoll to separatecells according to density is described by van Mourik and W. P.Zeizlmaker in Methods in Enzymology 121;174-182 (J. J. Langone, H. H.van Vunakis (eds.), Academic Press Inc., N.Y.). Gradients of varyingdensity of solutions of bovine serum albumin can also be used toseparate cells according to density. (See N. Moav and T. N. Harris, J.Immunol. 105, 1512, 1970; see also Raid, D. J. in Selected Methods inCellular Immunology, B. Misheli and S. Shiigi (eds.), W. H. Freeman andCo., San Francisco, 1987). Preferably separation is achieved bycentrifugation with Ficoll-Hypaque (Pharmacia, Uppsula, Sweden). Thefraction that is most enriched for desired antibody-producing cells canbe determined in a preliminary procedure using ELISA based assays toselect populations that may contain antibodies with the desired bindingspecificity. Alternatively or in addition, the fraction most enrichedfor the desired antibody can be determined by a functional assay.

In the assay the population of antibody producing cells suspected ofproducing antibodies with the desired binding specificity are suspendedin an appropriate medium before incorporation into the assay. Anappropriate medium for the assay will be one that provides at least theminimum requirements for short-term maintenance of cellular integrityand cellular structures, such as an isotonic buffer. Preferably thismedium is immune cell medium comprising Roswell Park Memorial Institutemedium (RPMI)+10% foetal bovine serum; 50 μM 2-β-mercaptoethanol; 2 mMglutamine; 20 mM Hepes; and 1× Penicillin and Streptomycin.

Under such conditions the antibody producing cells produce and secreteantibodies. Antibody producing cells are diluted within the medium to adensity which allows selection of an individual or small number ofantibody producing cells. If it is unclear which cell is responsible forthe activity indicated by the assay, or in order to confirm theactivity, the selected cell(s) may be retested for their ability toproduce antibodies with the desired binding specificity.

The antigen for use in the assay may be, as described above, anysubstance to which an antibody can be produced including proteins,glycoproteins, carbohydrates and whole cells, such as tumor cells ortransfected cells expressing the antigen on the surface. In one examplethe antigen is known and available in a pure form and is coated on thesurface of erythrocytes or other particles such as beads forincorporation into the assay. A number of methods for coating particleswith antigens are known to those skilled in the art. These includechromic chloride or water soluble carbodiimide. In one embodiment, abiotin/streptavidin coupling system is used to couple antigen toerythrocytes, the methods for which are described in detail inWO92/02551.

In another example the antigen is coupled to commercially availablebeads (for example as can be obtained from New England Biolabs). Antigencan be conjugated to beads using a number of different methods,preferably via direct conjugation to activated beads or via biotin tostreptavidin-coupled beads. Preferably these beads are magnetic for easeof handling.

In another example, particularly when the antigen loses desirableepitopes upon biotinylation, the antigen is coupled to the surface of aparticle via a polyclonal antibody that binds the antigen. To preparethe antigen-polyclonal antibody-particle conjugate, the polyclonalantibody is first conjugated to the surface of a particle, such as abead using any suitable method, such as via biotin tostreptavidin-coupled beads. The polyclonal antibody-particle conjugateis then incubated with an excess of antigen to allow binding of thepolyclonal antibody to the antigen. The antigen-polyclonalantibody-particle conjugate is then separated from unbound antigen, forexample by centrifugation, and incorporated into the assay. Thepolyclonal antibody for use in the conjugate may be produced using anysuitable method known in the art, using the desired antigen asimmunogen, in any suitable species. The polyclonal antibody may be awhole IgG or a fragment thereof such as a Fab′, F(ab′)₂ or Fab fragment.Fragments may be produced using any method known in the art, for exampleby pepsin or papain digestion. It is important that the polyclonalantibody used in the conjugate is not recognized by the labeledanti-antibody antibody used in the assay to detect the antibodiesproduced by the antibody producing cells. For example where the antibodyproducing cells are from rabbit, the labeled anti-antibody antibody maybe an anti-rabbit anti-Fc antibody and the polyclonal antibody used inthe conjugate should be an antibody from a species other than rabbit,for example goat, or if the antibody is from rabbit it should be afragment lacking the Fc region, for example a Fab′, F(ab′)₂ or Fabfragment.

In another example, particularly when the antigen is difficult to purifyor loses desired epitopes upon biotinylation the antigen is expressed onthe surface of a cell. Such cells may be those that naturally expressthe antigen on their surface or a transfected cell expressing theantigen on its surface. Such cells may include but are not limited tomammalian cells, immunomodulatory cells, lymphocytes, monocytes,polymorphs, T cells, tumor cells, yeast cells, bacterial cell,infectious agents, parasites, plant cells, transfected cells such asNS0, CHO, COS, 293 cells. Transfection of cells such as NS0, CHO, COSand 293 cells can be achieved by any method known in the art including,electroporation and nucleofection.

In a further example the antigen source is any cell that it would bedesirable to isolate antibodies to. Such cells may include but are notlimited to mammalian cells, immunomodulatory cells, lymphocytes,monocytes, polymorphs, T cells, tumor cells, yeast cells, bacterialcell, infectious agents, parasites and plant cells.

The antibody producing cells and the antigen are incorporated into theassay at an appropriate concentration which can be determinedempirically for example as described in the examples hereinafter. Theantibody producing cells are at sufficiently low density that they arewell separated allowing identification and isolation of the antibodyproducing cell producing antibodies of the desired specificity. Theantigen will be present in excess and preferably the antigen is in a10-1,000 fold excess over the antibody producing cells.

In order to identify antibodies that bind to the selected antigen,labeled anti-antibody antibodies are incorporated into the assay. Saidantibodies will bind to all antibodies produced by the antibodyproducing cells, regardless of their binding specificity. Suchantibodies are easily produced by one skilled in the art or are readilyavailable commercially. Preferably the anti-antibody antibodies areanti-Fc antibodies. In one embodiment of the present invention theantibody producing cells are from rabbits and the labeled anti-Fcantibodies are goat anti-rabbit anti-Fc antibodies.

The label conjugated to the anti-antibody antibodies is any label thatcan be detected in the assay by any suitable method known in the art.Many different conjugates are available for labeling the antibodies forexample, chemiluminescent, enzyme and fluorescent labels. Suchantibodies are easily produced by one skilled in the art or are readilyavailable commercially. Preferably the label is one that can be detectedby microscopy. In general in the various aspects of the inventiondescribed herein the label used is preferably a fluorescent label.Particular fluorescent labels are those which can be visualised bymicroscopy and can include but are not limited to Aqua, Texas-Red, FITC,rhodamine, rhodamine derivatives, fluorescein, fluorescein derivatives,cascade blue, Cy5 and phycoertythrin. Preferably said label is thefluorescent conjugate, fluorescein isothiocyanate The labeledanti-antibody antibody is used in the assay at a concentration at whichit is possible to distinguish cells producing antibodies that bind tothe selected antigen from those cells that do not. The optimalconcentration can be determined empirically by one skilled in the art byvarying the concentration of labeled anti-antibody antibody. In oneexample the labeled antibody is a fluorescent labeled antibody and isused at a concentration that is not so low that no fluorescence can bedetected and not so high that there is high background fluorescence.Preferably the fluorescent labeled anti-antibody antibody is in excesssuch that it binds all antibodies produced by the antibody producingcell without causing excessive background fluorescence.

To identify antibody producing cells producing antibodies which bind tothe selected antigen the assay mixture comprising a population ofantibody producing cells, the antigen and labeled anti-antibody antibodyis incubated in the medium described above to allow binding to takeplace. Optimal incubation times and temperatures can be determinedempirically by one skilled in the art. Incubation will take place in anysuitable container such as a microscope slide at any suitabletemperature for example between 4° C. or about and 37° C. or about, forany suitable length of time for example between 5 minutes or about and 5hours or about. Preferably the incubation of the assay mixture takesplace on a microscope slide at 37° C. for up to 1 hour.

The labeled anti-antibody antibody is detected using any appropriatemethod known in the art. Preferably the labeled anti-antibody antibodyis detected using a microscope. More preferably the anti-antibodyantibody is conjugated to a fluorescent label and the fluorescence isvisualised using an inverted microscope equipped with a mercury vapourUV lamp with an appropriate filter set. Preferably the filter set is afluorescein filter set.

Antibody producing cells which produce an antibody which binds to theselected antigen are identified by the increased concentration oflabeled anti-antibody antibodies surrounding the cell. Those antibodyproducing cells producing antibodies which do not bind to the antigenwill not be surrounded by an increased concentration of labeledanti-antibody antibodies. High yielding antibody producing cells areidentified as those where the localised increase in anti-antibodyantibody concentration appears most quickly.

The antibody producing cell may then be isolated directly from the assayusing standard micromanipulation techniques such as a fine glass pipetteand a micromanipulator. Antibodies can be synthesized directly orindirectly from the isolated antibody producing cell. Direct synthesiscan be achieved by culturing the isolated antibody producing cell in anappropriate medium. If the assay is used to identify a high yieldingantibody producing cell the cell will be cultured under appropriateconditions to clonally reproduce this high yielding cell.

Indirect synthesis can be achieved by isolating the genes encoding theantibodies or parts thereof and expressing them in a host cell. Theentire genes may be cloned or the variable regions or portions thereofwhich confer the desired specificity of the antibody may be cloned andused to produce recombinant antibodies. Recombinant antibodies can takeseveral different forms and include intact immunoglobulins, chimericantibodies, humanised antibodies and antigen binding fragments such asFv, Fab, Fab′ and F(ab′)₂ fragments, and any derivatives thereof, suchas single chain Fv fragments. The methods for creating these antibodymolecules are well known in the art (see for example, Boss, U.S. Pat.No. 4,816,397; Shrader, WO 92/02551; Ward et al., 1989, Nature, 341,544; Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 3833;Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 6851; Riechmannet al., 1988, Nature, 322, 323; Bird et al, 1988, Science, 242, 423).The types of expression systems available to produce these antibodymolecules include bacterial, yeast, insect and mammalian expressionsystems, the methods for which are well known in the art (Verma et al.,1998, Journal of Immunological Methods, 216, 165-181).

Antibodies obtained according to the invention may be used withoutfurther modification, or if desired following modification includingconjugation to one or more reporter or effector molecules, for anysuitable diagnostic or therapeutic purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A homogeneous fluorescence assay comprising rabbit B cells, sheepred blood cells coated with antigen and Goat anti-Rabbit IgG Fc specificFITC conjugate. Assay visualised using an inverted microscope equippedwith a mercury vapour UV lamp and fluorescein filter set. Magnification×8.

(a) Phase image of assay.

(b) Fluorescence image of assay. Fluorescence localised around B cellssecreting antigen specific antibodies

FIG. 2 A homogeneous fluorescence assay comprising rabbit B cells,magnetic beads coated with antigen and Goat anti-Rabbit IgG Fc specificFITC conjugate. Assay visualised using an inverted microscope equippedwith a mercury vapour UV lamp and fluorescein filter set. Magnification×20.

(a) Phase image of assay.

(b) Fluorescence image of assay. Fluorescence localised around B cellssecreting antigen specific antibodies

FIG. 3 ELISA detection of specific antigen binding of antibodiesproduced in CHO cells.

FIG. 4 A homogeneous fluorescence assay comprising rabbit B cells,transfected COS-1 cells expressing antigen on their surface and Goatanti-Rabbit IgG Fc specific FITC conjugate. Assay visualised using aninverted microscope equipped with a mercury vapour UV lamp andfluorescein filter set. Magnification ×8

(a) Phase image of assay.

(b) Fluorescence image of assay. Fluorescence localised around B cellssecreting antigen specific antibodies.

FIG. 5 A homogeneous fluorescence assay comprising rabbit B cells,transfected CHO cells expressing antigen on their surface and Goatanti-Rabbit IgG Fc specific FITC conjugate. Assay visualised using aninverted microscope equipped with a mercury vapour UV lamp andfluorescein filter set. Magnification ×8.

(a) Phase image of assay.

(b) Fluorescence image of assay. Fluorescence localised around B cellssecreting antigen specific antibodies

The following examples are offered by way of illustration, and not byway of limitation.

The following abbreviations are used in the examples:

ICM—immune cell medium (RPMI+10% foetal bovine serum; 50 μM2-β-mercaptoethanol; 2 mM glutamine; 20 mM hepes; and 1× penicillin andstreptomycin)

RPMI—Roswell Park Memorial Institute medium

PBS—Phosphate buffered saline

EXAMPLE 1

Identification of Specific Antibody Producing B-cells Using AntigenCoated Sheep Red Blood Cells (SRBC)

Antigen Coating of SRBC

The coating of the SRBC (obtained from TCS Biosciences) was carried outby streptavidin linking the biotinylated antigen to the surface ofbiotin coated SRBC. The antigen coated SRBC were prepared on the day ofuse and stored 5% (v/v) in immune cell medium.

Identification of Antigen Specific Antibody Secreting B-Cells

The assay mix was set up in ICM and contained 10 μl of rabbit B cellscontaining 10-1,000 B cells from an ELISA positive population, 10 μlantigen coated SRBC (5% v/v) and 20 μl of Goat anti-Rabbit IgG Fcspecific FITC conjugate (Jackson ImmunoResearch) at variableconcentrations for each experiment (1:100, 1:200, 1:400 and 1:800). Theexperiments were set up to determine the optimal concentration of Goatanti-Rabbit IgG Fc specific FITC conjugate required for theidentification of antibody producing cells without excessive backgroundfluorescence.

This assay mix was then spotted (2-3 μl per spot), onto Sigmacote®treated ‘chamber’ slides and flooded with light paraffin oil. Slideswere incubated for 20-30 mins at 37° C. and examined using an invertedmicroscope equipped with a mercury vapour UV lamp and a fluoresceinfilter set. B cells, (plasma cells), secreting antigen specific IgGantibody were identified by a focal increase in fluorescence surroundingsaid cells. See FIG. 1. Using this method the optimal concentration forGoat anti-Rabbit IgG Fc specific FITC conjugate was found to be 1:400.Other B cells in the mixture, which did not secrete antigen specificantibodies, did not show surrounding fluorescence. In SRBC controlswhere no antigen was present on the surface, no B-cell localisedfluorescence was observed.

The B cells present within the fluorescent foci were then harvested intoEppendorf tubes using standard micro-manipulation apparatus, (EppendorfTransferman and CeilTram Vario) and the heavy and light chain variableregions of the antibody subsequently isolated by PCR.

EXAMPLE 2

Identification of Specific Antibody Producing B Cells Using AntigenCoated Beads

1 μM magnetic streptavidin coated beads, (New England Biolabs), wereused in all experiments.

Determination of Optimal Density for Bead Monolayer.

An aliquot of the bead stock was washed 3× in PBS to removepreservative, using a magnet, and resuspended in the same volume ofimmune cell medium, (ICM), (RPMI+10% foetal bovine serum; 50 μM2-β-mercaptoethanol; 2 mM glutamine; 20 mM Hepes; and 1× Penicillin andStreptomycin).

Serial 2-fold dilutions of beads were prepared in ICM and used todetermine a dilution with a bead density that produced an even monolayerwhen the beads were spotted (2-3 ul per spot) onto Sigmacote treatedslides and overlaid with light paraffin oil. A final dilution of washedbeads of ⅛ was determined to be optimal.

Antigen Loading Onto Beads and Identification of Antigen Specific BCells.

50 μl aliquots of streptavidin coated beads were washed and resuspendedin 50 μl PBS. Each aliquot was incubated with different amounts of stockbiotinylated antigen (1 mg/ml) ranging from 0.1 μg up to 25 μg. Thesewere incubated for 1 h at room temperature with occasional manualshaking. The beads were then washed in PBS using a magnet andresuspended in 50 μl of ICM.

20 μl of the antigen loaded beads were then mixed with 40 μl of ELISApositive B cells; 60 μl of ICM; and 40 μl of a 1/400 dilution of a Goatanti-Rabbit IgG Fc specific FITC conjugate (Jackson ImmunoResearch).

This mixture was then spotted, (2-3 μl per spot), onto Sigmacote treated‘chamber’ slides and flooded with light paraffin oil. Slides wereincubated for 20-30 mins at 37° C. and examined using an invertedmicroscope equipped with a mercury vapour UV lamp and a fluoresceinfilter set.

B cells, (plasma cells), secreting antigen specific IgG antibody wereidentified by a focal increase in fluorescence around the B cell. SeeFIG. 2. Using this method 1 μg of biotinylated antigen per 50 μl of beadstock was determined as optimal for signal generation for thisparticular antigen. Other B cells in the mixture, which did not secreteantigen specific antibodies, did not show surrounding fluorescence. Incontrols, no B-cell localised fluorescence was observed when the beadswere coated with another irrelevant antigen.

The B cells present within the fluorescent foci were then harvested intoEppendorf tubes using standard micro-manipulation apparatus, (EppendorfTransferman and CellTram Vario) and the heavy and light chain variableregions of the antibody from one of the cells subsequently isolated byPCR. A recombinant chimeric IgG (human constant regions) was produced bytransient expression in CHO cells. Transfections of CHO cells wereperformed using the lipofectamine procedure according to manufacturer'sinstructions (InVitrogen, catalogue no. 18324). Specific binding of thIgG to antigen was confirmed by ELISA (FIG. 3).

EXAMPLE 3

Identification of Specific Antibody Producing B-Cells Using SurfaceExpression of Antigen on COS-1 Cells

Transient Expression of Antigen on COS-1 Cells

COS-1 cells transiently expressing the selected antigen were suspendedin Immune cell media. Cell density was altered to 2×10⁷ cells per ml.

Identification of Antigen Specific Antibody Secreting B-Cells

The assay mix was set up in ICM and contained 40 μl ELISA positive Bcells, 40 μl of Goat anti-Rabbit IgG Fc specific FITC conjugate (JacksonhnmunoResearch) at 1:400 dilution and 40 μl of COS-1 cell suspension.

This assay mix was then spotted (2-3 ul per spot), onto Sigmacotetreated ‘chamber’ slides and flooded with light paraffin oil. Slideswere incubated for 40 mins at 37° C. and examined using an invertedmicroscope equipped with a mercury vapour UV lamp and a fluoresceinfilter set.

B cells, (plasma cells), secreting antigen specific IgG antibody wereidentified by a focal increase in fluorescence surrounding the B cells.See FIG. 4. Other B cells in the mixture, which did not secrete antigenspecific antibodies, did not show surrounding fluorescence. In COS-1cell controls where no antigen was present on the surface, no B-celllocalised fluorescence was observed.

The B cells present within the fluorescent foci were then harvested intoEppendorf tubes using standard micro-manipulation apparatus, (EppendorfTransferman and CellTram Vario) and the heavy and light chain variableregions of the antibody subsequently isolated by PCR.

EXAMPLE 4

Identification of Specific Antibody Producing B-Cells Using SurfaceExpression of Antigen on Chinese Hamster Ovary (CHO) Cells

Transient Expression of Antigen on CHO Cells

CHO cells transiently expressing the selected antigen were suspended inImmune cell media. Cell density was altered to 2×10⁷ cells per ml.

Identification of Antigen Specific Antibody Secreting B-Cells

The assay mix was set up in ICM and contained 40 μl ELISA positive Bcells, 40 μl of Goat anti-Rabbit IgG Fc specific FITC conjugate (JacksonImmunoResearch) at 1:400 dilution and 40 μl of CHO cell suspension.

This assay mix was then spotted (2-3 μl per spot), onto Sigmacotetreated ‘chamber’ slides and flooded with light paraffin oil. Slideswere incubated for 40 mins at 37° C. and examined using an invertedmicroscope equipped with a mercury vapour UV lamp and a fluoresceinfilter set.

B cells, (plasma cells), secreting antigen specific IgG antibody wereidentified by a focal increase in fluorescence surrounding the B cells.See FIG. 5. Other B cells in the mixture, which did not secrete antigenspecific antibodies, did not show surrounding fluorescence. In CHO cellcontrols where no antigen was present on the surface, no B-celllocalised fluorescence was observed.

The B cells present within the fluorescent foci were then harvested intoEppendorf tubes using standard micro-manipulation apparatus, (EppendorfTransferman and CellTram Vario) and the heavy and light chain variableregions of the antibody subsequently isolated by PCR.

1. A homogeneous assay for identifying a cell that produces an antibodythat binds to a selected antigen comprising: providing a population ofantibody producing cells; incubating the population of antibodyproducing cells with a selected antigen and a labeled anti-antibodyantibody, wherein the anti-antibody antibody distinguishes cells thatproduce an antibody that binds to the selected antigen from cells thatdo not; and identifying a cell that produces an antibody that binds tothe selected antigen.
 2. The assay of claim 1 where the antigen iscoupled to an erythrocyte.
 3. The assay of claim 1 where the antigen iscoupled to a bead.
 4. The assay of claim 3 where the antigen is coupledto a bead via a polyclonal antibody.
 5. The assay of claim 4 where thepolyclonal antibody is an antibody fragment.
 6. The assay of claim 5where the polyclonal antibody is an antibody Fab, Fab′ or F(ab′)₂fragment.
 7. The assay of claim 1 where the antigen is expressed on thesurface of a cell.
 8. The assay of claim 7 where the cell is atransfected cell.
 9. The assay of claim 7 where the cell is a tumorcell.
 10. The assay of claim 1 where the antigen is an infectious agent.11. The assay of claim 1 where the labeled anti-antibody antibody is ananti-Fc antibody.
 12. The assay of claim 1 where the labeledanti-antibody antibody is labeled with a fluorescent conjugate.
 13. Theassay of claim 12 where the fluorescently-labeled anti-antibody antibodyis labeled with FITC.
 14. The assay of claim 13 where the FITC labeledanti-antibody antibody is an anti-Fc antibody.
 15. The assay of claim 1where the antibody producing cells are B cells, plasma cells,plasmablasts, activated B cells or memory B cells.
 16. The assay ofclaim 1 where the cell that produces an antibody is a hybridoma cell ora mammalian cell engineered to express antibodies.
 17. A cell thatproduces an antibody identified by the assay of claim
 1. 18. A methodfor producing an antibody that binds to a selected antigen comprising:providing a population of antibody producing cells; incubating thepopulation of antibody producing cells with a selected antigen and alabeled anti-antibody antibody, wherein the anti-antibody antibodydistinguishes cells that produce an antibody that binds to the selectedantigen from cells that do not; identifying a cell that produces anantibody that binds to the selected antigen; isolating the identifiedcell that produces an antibody; and optionally synthesizing an antibodyor antibody fragment from the isolated cell that produces an antibody.19. A cell that produces an antibody isolated by the method of claim 18.20. An antibody produced by the method of claim 18.